Inhibition of tau-tau-association

ABSTRACT

A composition and use thereof for the prophylaxis or treatment of Alzheimer&#39;s disease, motor neurone disease, Lewy body disease, Pick&#39;s disease or progressive supranuclear palsy. The composition includes an agent which modulates or inhibits tau-tau association and which does not inhibit tau-tubulin binding. In the method the agent is administered to an individual in order to achieve the desired therapeutic effect. Preferably the agent which is administered is a phenothiazine compound.

This application is a continuation application of pending U.S.application Ser. No. 11/175,153, filed Jul. 7, 2005 (of which the entiredisclosure of the pending prior application is hereby incorporated byreference) now abandoned, which is a continuation application of U.S.application Ser. No. 10/107,181, filed Mar. 28, 2002, now U.S. Pat. No.6,953,794 (of which the entire disclosure of the pending, priorapplication is hereby incorporated by reference), which is a divisionalapplication of Ser. No. 08/913,915, filed Dec. 12, 1997, now U.S. Pat.No. 6,376,205, granted Apr. 23, 2002, which is the national stage ofPCT/EP96/01307, filed Mar. 25, 1996.

The present invention relates to novel methods for the detection ofsubstances capable of modulating or inhibiting pathological tau-tauprotein association and pathological neurofilament aggregation. Themethods of the present invention are particularly useful in screeningsubstances for the prophylaxis and treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the most common single cause of dementia inlate life (Livingstone (1994) The scale of the problem. In: Dementia(eds. Burns and Levy) Chapman & Hall, London, pp. 21-35). Individualswith Alzheimer's disease are characterised by progressive dementia thatpresents with increasing loss of memory, disturbances in judgement,perception is and speech, and global intellectual deterioration (Rothand Iversen (1986) Brit. Med. Bull., 42. (special volume)).

The major pathological hallmarks of Alzheimer's disease are senileplaques and neurofibrillary tangles, both of which contain pairedhelical filaments (PHFs) of which the microtubule associated protein tauis a constituent (Wischik et al. (1988) Proc. Natl. Acad. Sci. USA, 85,4506-4510). Plaques also contain β-amyloid fibrils derived from an asyet undefined abnormality in the processing of the amyloid precursorprotein (APP; Kang et al. (1987) Nature, 325, 733-736).

Studies of Alzheimer's disease have pointed to loss of the normalrnicrotubule associated protein tau (Mukaetova-Ladinska et al. (1993)Am. J. Pathol., 143, 565-578; Wischik et al. (1995a) Neurobiol. Ageing,16: 409-417; Lai et al. (1995b) Neurobiol. Ageing, 16: 433-445),accumulation of pathological paired helical filaments (PHFS;Mukaetova-Ladinska et al. (1993), loc. cit.; Harrington et al. (1994a)Dementia, 5, 215-228; Harrington et al. (1994b) Am. J. Pathol., 145,1472-1484; Wischik et al., (1995a), loc. cit.) and loss of synapses inmid-frontal cortex (Terry et al. (1991) Ann. Neurol., 30, 572-580) asstrong discriminatory markers for cognitive impairment. Loss of synapses(Terry et al. loc. cit.) and loss of pyramidal cells (Bondareff et al.(1993) Arch. Gen. Psychiatry, 50, 350-356) are both correlated withmorphometric measures of tau-reactive neurofibrillary pathology, andthis correlates at the molecular level with an almost completeredistribution of the tau protein pool from soluble to polymerised form(PHFs) in Alzheimer's disease (Mukaetova-Ladinska et al. (1993), loc.cit.; Lai et al. (1995), loc. cit.). A possible explanation for thesechanges is that the pathological redistribution of tau protein into PHFscauses a failure of axonal transport in cortico-cortical associationcircuits through failure to maintain axonal tubulin in the polymerisedstate within pyramidal cells (Wischik et al. (1995a), loc. cit.; Wischiket al. (1995b) Neurobiol. Ageing, in press; Wischik et al (1995c)Structure, biochemistry and molecular pathogenesis of paired helicalfilaments in Alzheimer's disease. Eds. A. Goate and F. Ashall, in press;Lai et al., (1995), loc. cit.). A resulting failure of transport ofsynaptic constituents from projection soma to distant associationneocortex would lead to synaptic loss and cognitive impairment. Furtherfactors include the direct toxicity of PHF accumulation in pyramidalcells (Bondareff et al., (1993), Arch. Gen. Psychiat. 50: 350-356;(1994), J. Neuropath. Exp. Neurol. 53: 158-164), and the possible directtoxicity of truncated tau accumulation impairing cellular function (Menaet al. (1991), J. Neuropath. Exp. Neurol. 50: 474-490).

Although studies of molecular pathogenesis in model systems haveemphasised the neurotoxic role of β-amyloid accumulation (reviewed inHarrington and Wischik (1994) Molecular Pathobiology of Alzheimer'sdisease. In: Dementia (eds. A. Burns and R. Levy). Chapman & HallLondon, pp. 211-238), the evidence linking β-amyloid deposition directlywith cognitive impairment in humans is weak. It is more likely thataltered processing of APP is only one of several possible factors whichmight initiate altered processing of tau protein. Other initiatingfactors include unknown processes associated with apoE4 (Harrington etal. (1994b), loc. cit.), trisomy of chromosome 21 (Mukaetova-Ladinska etal. (1994) Dev. Brain Dysfunct. 7: 311-329), and environmental factors,such as prolonged exposure to sub-toxic levels of aluminium (Harringtonet al. (1994c) Lancet, 343, 993-997). Distinct etiological factors areable to initiate a common pattern of disturbance in tau proteinprocessing which includes: C-terminal truncation at Glu-391, formationof PHF tau polymers, loss of soluble tau, and accumulation of abnormallyphosphorylated tau species (Wischik et al (1996) Int. Rev. Psychiat., inpress).

The fragment of the microtubule-associated protein tau which has beenshown to be an integral constituent of the protease-resistant corestructure of the PHF is a 93/95 amino acid residue fragment derived fromthe microtubule binding domain of tau (Wischik et al. (1988), loc. cit.;Kondo et al. (1988) Neuron, 1, 827-834; Jakes et al. (1991) EMBO J., 10,2725-2729; Novak et al. (1993) EMBO J., 12, 365-370). Tau protein existsin 6 isoforms of 352-441 amino acid residues in the adult brain (Goedertet al. (1989) Neuron, 3, 519-526). In general structure the tau moleculeconsists of an extensive N-terminal domain of 252 residues, whichprojects from the microtubule, a tandem repeat region of 93-125 residuesconsisting of 3 or 4 tandem repeats and which is the microtubule bindingdomain, and a C-terminal tail of 64 residues. Each tandem repeat iscomposed of a 19 residue tubulin binding segment, and 12 residue linkersegment (Butner and Kirschner (1991) J. Cell Biol., 115, 717-730; FIG.1). The major tau constituent which can be extracted from enrichedprotease-resistant core PHF preparations is a 12 kDa fragment derivedfrom both 3- and 4-repeat isoforms, but restricted to the equivalent of3 tandem repeats regardless of isoform (Jakes et al., loc. cit.; FIG.2). The N- and C-terminal boundaries of the fragment define the preciseextent of the characteristic protease-resistant core PHF tau unit. It isphase-shifted by 14/16 residues with respect to the binder/linkerorganisation of the normal molecule defined by Butner and Kirschner,loc. cit., FIG. 1) and is C-terminally truncated at Glu-391, or at ahomologous position in the third repeat of the 4-repeat isoform (Novaket al. (1993), loc. cit.; FIGS. 3A, 3B and 3C). A monoclonal antibody(mAb 423) is available which specifically recognises this C-terminaltruncation point, and histological studies using this antibody haveshown the presence of tau protein C-terminally truncated at Glu-391 atall stages of neurofibrillary degeneration (Mena et al. (1995) ActaNeuropathol., 89, 50-56; Mena et al. (1996) Acia Neuropathol. (inpress)). Thus, a possible post-translation modification implicated inPHF assembly is abnormal proteolysis.

Methods have been developed which permit discrimination between severaltau pools found in AD brain tissues: normal soluble tau, phosphorylatedtau, and protease-resistant PHFs (Harrington et al. (1990), (1991),(1994a), loc. cit.). These methods have been deployed in studies ofsevere AD and Down's Syndrome (Mukaetova-Ladinska et al. (1993; 1995),loc. cit.), in prospectively assessed cases at early stage AD (Wischiket al. (1995a), loc. cit.; Lai et al. (1995), loc. cit.) and cases withother neuropathological diagnoses including senile dementia of the Lewybody type and Parkinson's disease (Harrington et al. (1994a), (1994b),loc. cit.). The overall PHF content in brain tissue distinguishesunambiguously between patients with and without dementia of theAlzheimer type. There is overall a 19-fold difference in PHF content,and in temporal cortex the difference reaches 40-fold. Furthermore,apolipoprotein E genotyping of the cortical Lewy body cases showed thatthe frequency of the E4 allele was raised to a similar extent to thatseen in AD. Therefore, the presence of the E4 allele cannot be the solecause of the characteristic tau pathology of AD, since this was not seenin the Lewy body cases (Harrington et al. (1994b), loc. cit.).

A further parameter which distinguishes cases with and without AD is theamount of normal soluble tau protein. Although tau levels are higher inwhite matter than in grey matter, as expected for an axonal microtubuleassociated protein, the amount found in grey matter also reflectsafferent axonal innervation. In AD, there is a substantial loss ofnormal soluble tau protein which affects all brain regions uniformly(Mukaetova-Ladinska et al. (1993), loc. cit.). The molecular basis ofthis uniform decline is not known, and cannot be explained by reducedtau mRNA (Goedert et al. (1988) Proc. Natl. Acad. Sci, USA, 85,4051-4055). The net effect the two processes of accumulation of PHFs andloss of soluble tau is an anatomical redistribution of the tau proteinpool, from white matter predominant to grey matter predominant, and fromfrontal predominant to temporo-parietal prodominant.

The global extent of tau protein redistribution in AD can be appreciatedfrom the data shown in FIG. 4, where total free and PHF-bound tau poolsare compared. Whereas in controls, 97% of the tau protein pool is in thesoluble phase, in AD 87% of the tau protein pool is to be found in theinsoluble phase, almost entirely in a form truncated and polymerisedinto PHFs (Mukaetova-Ladinska et al. (1993), loc. cit.). A study ofearly stage AD in cases prospectively assessed by the clinicaldiagnostic instniment CAMDEX (Roth et al. (1986) Brit. J. Psych., 149,698-709) and graded post-mortem by the staging criteria of Braak andBraak (1991), Acta Neuropathol. 82, 239-259) demonstrated that the lossof soluble tau is diredtly related to the tangle couni and to the extentof PHF accumulation (Lai et al. (1995), loc. cit.).

Although abnormally phosphorylated tau has been considered a possiblePHF precursor (Lee et. al. (1991) Science, 251, 675-678; Goedert et al.(1994), in Microtubules (Hyams and Lloyd, eds.) pp. 183-200. John Wiley& Sons, NY), normal tau has been found to be phosphorylated at many ofthe sites previously considered abnormally phosphorylated inPHF-associated tau protein (Matsuo et al. (1994) Neuron, 13, 989-1002).In the study of early stage AD, insoluble hyperphosphorylated tauspecies were first seen after appreciable tau redistribution into PHFshad occurred (Lai et al., 1995; FIG. 5). There was no evidence ofselective accumulation of phosphorylated species prior to the appearanceeither of PHFs, or of neurofibrillary tangles (Lai et al., (1995), loc.cit.). Likewise, there was no evidence that phosphorylated tau feedsinto the total PHF-bound pool during progression of pathology (Lai etal. (1995), loc. cit.). Phosphorylation of tau protein, insofar as it isabnormal, appears to be a secondary process affecting about 5% of PHFsat any stage of pathology (Wischik et al. (1995a), (1995c), oc. cit.).

Studies of early stage Alzheimer's disease also showed that the rate oftransfer of soluble tau into PHFs is geometric with respect to the PHFlevel, with a progressive increase in the rate of incorporation athigher ambient levels of PHFs (Lai et al. (1995), loc. cit.; FIG. 6B).Furthermore, the observed rate of loss of soluble tau with progressionof pathology is not enough to account entirely for the observed rate ofaccumulation of PHFs. Progressively more new tau synthesis is induced asthe ambient level of soluble tau falls below 580 pmol/g, and this toofeeds into PHF assembly (FIG. 6A). The rate of PHF assembly is thereforenot determined by the state or concentration of the soluble precursor,which appears to be entirely normal even in AD (Wischik et al. (1995a),(1995b), loc. cit.). Rather, the rate of transfer of soluble tau intoPHFs is determined by the ambient level of PHF-tau, suggesting that thecritical post-translational modification responsible for PHF assemblyoccurs at the point of incorporation of tau into the PHF.

A likely explanation for these findings is that tau protein undergoes aninduced conformational change at the point of incorporation into thePHF, which is associated with the half-repeat phase shift in the tandemrepeat region that has been documented previously (Novak et al. (1993),loc. cit.). This conformational change could expose a high affinity taucapture site which permits the capture and induced conformationalmodification of a further tau molecule, and so on. The criticalconformational change in tau protein which determines the rate of PHFassembly would not then need to be a chemical modification of solubletau, hut an induced conformational change which is produced by thebinding of tau protein to a pathological substrate. The process could beinitiated by non-tau proteins, such as a product of APP metabolism(Caputo et al. (1992) Brain Res., 597, 227-232), a modifiedmitochondrial protein (Wallace (1994) Proc. Natl. Acad. Sci. USA, 91,8739-8746), etc. Once tau capture had been initiated, the process couldcontinue provided the rate of further tau capture exceeded the rate ofdegradation of the pathological tau complex. Degradation could belimited by the fact that the core tau complex of the PHF is resistant toproteases (Wischik et al. (1988), loc, cit.; Jakes et al. loc. cit.).Such a process, an “amyloidosis of tau protein”, could be initiated andprogress geometrically without any intervening chemical modification ofsoluble tau protein, as commonly supposed.

FIG. 7 schematically depicts the transformation of tau protein into PHFsin Alzheimer's disease. The major protein constituent of the PHF core isa form of tau protein which is truncated down to a 93 residue fragmentwhich encompasses a phase-shifted version of the tandem repeat region ofthe tau molecule which normally functions as the microtubule bindingdomain. The assembly of the PHF can be envisaged as occurring as aresult of a repetitive sequence of events in which pathological tau-taubinding plays a pivotal role. This binding of free tau is favoured at aphysiological concentration only in the asymmetrical case in which onetau molecule has already undergone pathological capture (e.g. to aproduct of APP metabolism (Caputo et al. (1992) Neurobiol. Ageing, 13,267-274), or an altered mitochondrial protein (Jancsit et al. (1989)Cell Motil. Cytoskel., 14, 372-381; Wallace, loc. cit.), and further taubinding is enhanced by partial proteolytic processing of the capturedspecies leaving only the truncated tau unit. Once a full-length ortruncated unit binds a full-length molecule, partial proteolyticprocessing of the pathological complex results in the production of adimer of core tau units, with loss of N- and C-terminal domains of thepreviously intact molecule(s). The limits of proteolytic processing aredetermined by the region of tau-tau association, which correspondsprecisely to the minimal protease-resistant tau unit we have described(Novak et al. (1993), loc. cit.); see (FIGS. 17A, 17B and 17C). However,the end result of this partial proteolysis is to reproduce the core tauunit, which is able to capture a further full-length tau molecule. Thisprocess can be repeated indefinitely. It requires two key steps tocontinue to the point of exhaustion of the available tau protein pool.The first is repeated capture of full-length tau by the truncated unit,the second is truncation of bound full-length tau to reproduce the coreunit.

So far, no reliable methods for the measurement of pathological tau-tauassociation are available and no substances capable of modulating orinhibiting pathological tau-tau association have been described.

The solution to the above technical problem is achieved by providing theembodiments characterised in the claims.

Accordingly, the present invention relates to methods for the detectionof agents capable of modulating or inhibiting pathological tau-tauassociation comprising contacting

-   -   a) a tau protein or a derivative thereof containing the tau core        fragment with    -   b) an agent suspected of being capable of modulating or        inhibiting tau-tau association and with    -   c) a labelled tau protein or a labelled derivative thereof        capable of binding to the tau protein of step a) or with a tau        protein or a derivative thereof which is distinct from the tau        protein of step a) and also capable of binding to the tau        protein of step a) and    -   d) detection of the tau-tau binding.

The modification of tau which is responsible for its polymerisation intoPHFs is propagated by a physical conformational change rather than anypreceding

chemical post-translational modification of tau. Surprisingly, it ispossible to transfer this modification which is induced in vivo at thepoint of pathological tau capture to the in vitro method according tothe above process by initial tau binding to a solid phase. Tau isolatedfrom the brain of the rat neonate was entirely unable to bind to thecore tau unit of the PHF (FIG. 14; POTr). But neonatal tau which hadbeen previously bound passively to solid phase matrix, was induced tobind unmodified full-length tau protein with an identical high affinityto that demonstrated with the core tau unit (FIGS. 15 & 16). Thus, thecritical factor required to convert a species of tau incapable ofpathological binding, into a species able to capture a further taumolecule with high affinity, is the conformational change induced bypassive binding of neonatal tau to the solid phase substrate. Thisdemonstrated that the exposure of the high affinity tau capture sitecould be induced physically by the conformational change that occursupon binding of tau to a suitable substrate, and does not require anyother chemical modification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents tau protein binding to microtubules.

FIG. 2 shows a schematic representation of tau protein isoforms, whereontau fragments and the positions of epitopes for mAb are shown.

FIG. 3 shows the N-terminal sequence analysis of distinct peptidesreleased from core PHF preparations.

FIG. 4 shows the total (normal and PHF) tau protein content in controlsand Alzheimer's disease.

FIG. 5 shows the changes in soluble tau, phosphorylated tau, and tanglecount during early stages of Alzheimer's disease.

FIGS. 6A and 6B respectively show the calculated rates of transfer ofnew tau synthesis into the soluble tau pool, and of soluble tau into thePHF bound pool at early stages of Alzheimer's disease.

FIG. 7 shows a hypothetical scenario for transformation of tau proteininto PHFs in Alzheimer's disease showing autocatalytic cycles ofimmobilisation, truncation, captured, partial proteolytic degradationand further capture.

FIG. 8 shows a Tau binding assay configuration in which binding of twotruncated units is measured—a species terminating at Ala390 (“a”) and asecond truncated tau species terminating at Glu-391 (“e”).

FIG. 9 shows the binding of species “e” to “a” in phosphate bufferednormal saline (“normal”), distilled water (“water”) and sodium carbonatebuffer (“carbonate”, 50 mM, pH 9.6).

FIG. 10 shows the standard configuration for measurement of binding offull-length tau (“t”) to the truncated core tau unit previously boundpassively to the solid phase (“a”).

FIG. 11 shows the determination of Kd for binding of full-length tau(“T40”) to the truncated core tau unit terminating at Ala-390 (“a”),using mAb 499 to measure bound full-length human tau. The horizontalaxis on the upper graph shows the concentration of T40 used and thevertical axis shows mAb 499 immunoreactivity. Each binding curve isobtained at a plating concentration of “a” which is shown.

FIG. 12 shows the standard assay format shown in FIG. 10, with species“a” coated at 10 μg/ml and T40 added at the concentrations shown (range0-50 μg/ml), binding was measured at constant pH (pH 7.4), while varyingthe sodium chloride concentration.

FIG. 13 shows a similar experiment to that shown in FIG. 12, keeping thesodium chloride concentration constant at 137 mM, but varying the pH inthe range 3-10, with binding in physiological phosphate-buffered normalsaline (“PBS”, pH 7.4) and water (“0”) shown for comparison.

FIG. 14 shows typical sets of binding curves using the truncated coretau unit “a” in the solid phase, and incubating full length tau whichhas (“T40P”) or has not (“T40”) been phosphorylated. Fetal rat tau(“POTr”) when introduced in the aqueous phase is shown to be incapableof pathological binding to the core tau unit.

FIG. 15A shows a typical set of binding curves varying the concentrationof full-length tau (“T40”) and fetal tau (“PO Tau”) in the concentrationranges shown. The derived assymptotic Kd is shown in FIG. 15B.

FIG. 16 shows a comparison of Kd values in the tau-tau binding assayusing the species shown in the aqueous or solid phases.

FIGS. 17A, 17B and 17C show proteolytic digestion of aggregatedfull-length tau proteinas measured by immunoreactivity with variousstated mAb's. FIG. 17A shows full-length tau bound to dGA and incubatedwith Pronase. FIG. 17B shows full length tau which had self-aggregatedin the solid phase in the absence of dGA was digested similarly. FIG.17C shows the immunoreactivity of the digested fragments.

FIGS. 18A and 18B show accumulation of truncated tau by repetitive taucapture, beginning with the truncated tau fragment (dGA) in the solidphase, adding full-length recombinant human tau, and digesting withPronase, and repeating the addition and digestion. FIG. 18A showspronase digestion of the complex was associated with incrementalaccumulation of tau protein truncated at Glu-391 in the solid phasefollowing each digestion cycle. FIG. 18B shows binding of full-lengthtau was detected by the appearance of immunoreactivity for the Nterminus of tau which was entirely abolished by Pronase digestion.

FIGS. 19A and 19B shows relative tau-tau binding (vertical axis) in thepresence of increasing concentrations of prototype inhibitoryphenothiazines (horizontal axis).

FIG. 20 shows selective inhibition of tau-tau-binding by thionine.Truncated tau protein was used in both aqueous and solid phases of theassay as in FIG. 8 (filled circles). In the tau-tubulin assay,depolymerised tubulin was coated (open circles), and tau was incubatedwith it.

FIGS. 21A and 21B show nucleotide and predicted amino acid sequences ofa human tau protein isoform (SEQ ID NO: 4).

FIG. 22 shows amino acid and cDNA sequence of PHF-core tau unit (SEQ IDNO: 6) and the primers (SEQ ID NO: 7 and 8) used in construction of thepreferred core tau unit.

FIG. 23 shows the ranking of compounds by inhibition of tau-tauinteraction. Ranking is based on the standardised binding relative tothat seen in the absence of compound taken as the mean observed at 1 and10 μg/ml.

FIGS. 24A-24D show chemical structures of the compounds tested withvalues for standardised binding according to FIG. 17.

FIG. 25 shows a schematic representation of tau, MAP2 (adult form),MAP2C (juvenile form) and high molecular weight tau.

FIG. 26 shows sequence differences in the tandem repeat region of humantau (upper line; SEQ ID NO: 9) and human MAP2 (lower line; SEQ ID NO:10). Vertical arrows show the limits of the truncated PHF-core fragmentterminating atGlu-39 1, and the tubulin-binding segments are shownunderlined.

FIG. 27 shows the pIF2 expression vector which is an SV40-basedeukaryotic expression vector. Also shown are primers SEQ ID NO: 11 and12.

FIG. 28 shows mouse fibroblast 3T3 cells transfected with PIF2::T40,expressing full-length human tau protein (T40), immunolabelled by mAb7.51 (upper figure) and mAb 499 (lower figure).

FIG. 29 shows mouse fibroblast 3T3 cells transfected with PIF::dGAE,expressing the truncated PHF-core tau fragment terminating at Glu-391,immunolabelled with mAb 7.51.

FIG. 30 shows lipofectin/tau protein transfers into 3T3 cellstransfected with PIF2::T40. Relative cell survival is shown forapproximately equimolar concentrations of full-length (T40) andtruncated tau (dGAE), without (unshaded) or with shaded) thionine.

FIG. 31A shows reversal of truncated tau toxicity: the toxicity oftruncated tau transferred via lipofectin into 3T3 cells expressingfull-length tau is concentration dependent. Thionine (full-line)significantly reversed toxicity seen in the absence of thionine (brokenline). FIG. 31B shows a similar experiment in which full-length tau wastransferred via lipofectin into 3T3 cells expressing full-length.

According to the invention, the pathological binding which is reproducedin vitro had certain critical properties identical to those seen in thehuman brain. This is in particular that full-length tau protein (FIG.21, SEQ ID NO:4) bound to a core tau unit terminating at Ala-390 (FIGS.3A and 3C), and therefore lacking the Glu-391 needed for recognition bymonoclonal antibody 423, could be made to react with mAb 423 aftertreatment of the bound tau complex with the broad spectrum protease,Pronase, in a manner that depended quantitatively in the extent ofPronase digestion (FIGS. 17A, 17B and 17C. Digestion-dependent loss ofN-terminal tau immunoreactivity could be demonstrated to occur inparallel with the acquisition of the mAb 423 immunoreactivitycharacteristic of the core PHF (FIGS. 17A, 17B and 17C). Thus, theessential requirement needed for the creation of the tau unit isolatedfrom the core of the PHF, and produced in the brain in Alzheimer'sdisease is the pathological tau-tau interaction which had beenreproduced in vitro.

Further, repetitive cycles of binding of full-length tau to the core tauunit terminating at Ala-390, followed by treatment with Pronase, thenbinding of full-length tau and further Pronase digestion, and so on upto four cycles, was associated with progressive accumulation of tauC-terminally truncated at Glu-391 (FIG. 18A), and with progressivelyenhanced capacity to bind more full-length tau after each cycle (FIG.18B). This demonstrated that the essential role of proteolysis in themodel depicted in FIG. 7 is to prevent saturation, and hence facilitatesthe unlimited progressive transformation of soluble tau into thetruncated tau units of the core PHF.

Having shown that all the steps depicted in FIG. 7 could be reproducedin vitro, and that the critical requirement for progression of theprocess was the high affinity tau capture step, it is possible todemonstrate the use of the binding assay to find compounds able to blockthe high affinity tau-tau interaction. Competitive inhibition of 20%could be demonstrated when the most potent inhibitory compounds werepresent at 1:1 molar ratio with respect to tau, and further inhibitionwas found to be approximately linear in the range up to 10:1 molar ratio(FIG. 19).

Since the tandem repeat region functions as a whole, it is unexpectedthat it would be possible to demonstrate selective competitiveinhibition of pathological tau-tau binding without interference to thenormal binding of tau to tubulin via the same region of the molecule. Amethod of determining any possible interference, i.e. binding of tau ora derivative thereof to tubulin molecules, comprises contacting adepolymerised tubulin preparation, or preparation of taxol-stabilisedmicrotubules with an agent suspected of being capable of modulating orinhibiting pathological tau-tau association and a tau compound mentionedin above step c) followed by detection of the tau-tubulin binding.

The term “tau protein” refers to any protein of the tau protein familymentioned above and derivatives thereof. Tau proteins are characterisedas one family among a larger number of protein families which co-purifywith microtubules during repeated cycles of assembly and disassembly(Shelanski et al. (1973) Proc. Natl. Acad. Sci. USA, 70, 765-768), andknown as microtubule-associated-proteins (MAPs). The tau family inaddition is characterised by the presence of a characteristic N-terminalsegment which is shared by all members of the family, sequences of ˜50amino acids inserted in the N-terminal segment, which aredevelopmentally regulated in the brain, a characteristic tandem repeatregion consisting of 3 or 4 tandem repeats of 31-32 amino acids, and aC-terminal tail (FIG. 2).

In a preferred embodiment of the present invention the tau proteincomprises the amino acid sequence of FIG. 21 (SEQ ID NO: 5), referred toas “T40” (Goedert et al. (1989), Neuron 3: 519-526), or fragmentsthereof and comprising the form of the tau protein having 2 N-terminalinserts and 4 tandem repeats.

The term “tau core fragment” is defined in its most basic form as taufragment comprising a truncated tau protein sequence derived from thetandem repeat region which in the appropriate conditions is capable ofbinding to the tandem repeat region of a further tau protein with highaffinity. Ordinarily, preferred tau proteins, tau protein derivativesand tau protein core fragments have an amino acid sequence having atleast 70% amino acid sequence identity with the corresponding human tauprotein amino acid sequence (FIG. 21, SEQ ID NO: 5), preferably at least80% and most preferably at least 90% and are characterised in that theyare capable to bind to the human tau core fragment. A particularlyadvantageous embodiment of the assay method comprises the tau corefragment with the amino acid sequence shown in FIG. 22 (SEQ ID NO: 6;Novak et al., 1993). This recombinant tau peptide expressed by E. coliin vitro corresponds to species isolated from protease-resistantcore-PHF preparations (Wischik et al. (1988), loc. cit.; Jakes et al.(1991), loc. cit.). The term “tau core fragment” also includesderivatives thereof as described below and mentioned in FIGS. 25 and 26(SEQ ID NO: 9 and 10).

The terms “tau protein derivative” and “tau core fragment derivative”comprise fragments of naturally or non-naturally occurring tau proteinsand related proteins comprising at least partial amino acid sequencesresembling to the tandem repeat region of the tau proteins, i. e.proteins in which one or more of the amino acids of the natural tau orits fragments have been replaced or deleted without loss of bindingactivity. Examples of naturally occurring proteins with sequencesimilarity in the tandem repeat region are microtubule-associatedproteins (MAP2; FIGS. 25 and 26; SEQ ID NO: 9 and 10; Kindler and Garner(1994) Mol. Brain Res. 26, 218-224). Such analogues may be produced byknown methods of peptide chemistry or by recombinant DNA technology.

The terms “tau protein derivative” and “tau core fragment derivative”comprise derivatives which may be prepared from the functional groupsoccurring as side chains on the residues or the N- or C-terminal groups,by means known in the art. These derivatives may include aliphaticesters of the carboxyl groups, amides of the carboxyl groups by reactionwith ammonia or with primary or secondary amines, N-acyl derivatives offree amino groups of the amino acid residues formed with acyl moieties(e.g. alkanoyl or carbocyclic aroyl groups) or o-acyl derivatives offree hydroxyl groups (for example that of seryl- or threonyl residues)formed with acyl moieties.

The core PHF tau fragment may be isolated from AD brain tissues by themethod described in Wischik et al. (1988); (1995a), loc. cit.). Themethod depends on a series of differential centrifugation stepsconducted in empirically determined buffer and density conditions, thefinal critical centrifugation step being carried out in a continuoussucrose density gradient ranging between 1.05 and 1.18 in density and inthe presence of 10 μg/ml of Pronase, to produce a protease-resistantcore PHF-fraction at the interface with a high density caesium chloridecushion. Tau protein can be released from the core PHF as an essentiallypure preparation in the pH 5.5 supernatant (50 mmol, ammonium acetate)obtained after treating the PHF preparation with concentrated formicacid, lyophilisation, and sonication in pH 5.5 buffer.

Normal soluble tau can be isolated either from AD, control human braintissues, or from animal brain tissues, with a post-mortem delay of lessthan 3 hours, Microtubule proteins are obtained by three cycles oftemperature-dependent assembly-disassembly according to Shelanski et al.(1973, loc. cit.). Tau protein is purified from the thermostablefraction by gel filtration (Herzog and Weber (1978) Eur. J. Biochem.,92, 1-8). Alternatively, tau protein can be isolated by the procedure ofLindwall and Cole (1984; J. Biol. Chem., 259, 12241-12245) based on thesolubility of tau protein in 2.5% perchloric acid.

The production of tau proteins and fragments can further be achieved byconventional recombinant DNA technology which are within the skills ofan artisan in the field. Such techniques are explained further in theliterature, see e.g. Sambrook, Fritsch & Maniatis “Molecular Cloning. ALaboratory Manual” (1989) Cold Spring Harbor Laboratory, N.Y. andAusubel et al. “Current Protocols in Molecular Biology”, Green Publish.Association & Wiley Interscience.

Further, DNA molecules or fragments thereof encoding complete or partialtau proteins may be obtained with the polymerase chain reaction (PCR)technique. Primers encoding 3′ and 5′ portions of relevant DNA moleculesmay be synthesised for the tau protein of interest and can be utilisedto amplify the individual members of the tau protein family.

Preparation of tubulin proteins or fragments thereof are known in theart and are described e.g. by Slobada et al. (1976, in: Cell Mobility(R. Goldman, T, Pollard and J. Rosenbaum, eds.), Cold Spring Laboratory,Cold Spring Harbor, N.Y., pp 1171-1212). The DNA sequences and DNAmolecules may be expressed using a wide variety of host/vectorcombinations. For example, useful expression vectors may consist ofsegments of chromosomal, non-chromosomal and synthetic DNA sequences.Examples of such vectors are viral vectors, such as the various knownderivatives of SV40, bacterial vectors, such as plasmids from E. coli,phage DNAs, such as the numerous derivatives of phage λ, M13 and otherfilamentous single-stranded DNA phages, as well as vectors useful inyeasts, such as derivatives of the 2μ plasmid, vectors useful ineukaryotic cells more preferably vectors useful in animal cells, such asthose containing SV40, adenovirus and/or retrovirus derived DNAsequences.

As used herein, the term “DNA sequence” refers to a DNA polymer, in theform of a separate fragment or as a component of a larger DNA construct,which has been derived from DNA isolated at least once in substantiallypure form, i.e., free of contaminating endogenous materials and in aquantity or concentration enabling identification, manipulation, andrecovery of the sequence and its component nucleotide sequences bystandard biochemical methods, for example, using a cloning vector. Suchsequences are preferably provided in the form of an open reading frameuninterrupted by internal non translated sequences, or introns, whichare typically present in eukaryotic genes. However, it will be evidentthat genomic DNA containing the relevant sequences could also be used.Sequences of non-translated DNA may be present 5′ or 3′ from the openreading frame, where the same do not interfere with manipulation orexpression of the coding regions.

As used herein, the terms “expression vector” and “expression plasmid”refer to a plasmid comprising a transcriptional unit comprising anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, promoters or enhancers, (2) astructural or coding sequence which is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences. Structural elementsintended for use in various eukaryotic expression systems preferablyinclude a leader sequence enabling extracellular secretion of translatedprotein by a host cell. Alternatively, where recombinant protein isexpressed without a leader or transport sequence, it may include anN-terminal methionine residue. This residue may optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

The host cell used for the expression of DNA sequence may be selectedfrom a variety of known hosts. Examples for such hosts are prokaryoticor eukaryotic cells. A large number of such hosts are available fromvarious depositories such as the American Type Culture Collection (ATCC)or the Deutsche Sammlung für Mikroorganismen (DSM). Examples forprokaryotic cellular hosts are bacterial strains such as E. coli, B.subtilis and others. Preferred hosts are commercially availablemammalian cells such as mouse 3T3 cells, neuroblastoma cell lines suchas NIE-115, N2A, PC-12, or the SV40 transformed African Green monkeykidney cell line COS, etc.

The tau protein produced by fermentation of the prokaryotic andeukaryotic hosts transformed with the DNA sequences of this inventioncan then be purified to essential homogeneity by known methods such as,for example, by centrifugation at different velocities, by precipitationwith ammonium sulphate, by dialysis (at normal pressure or at reducedpressure), by preparative isoelectric focusing, by preparative gelelectrophoresis or by various chromatographic methods such as gelfiltration, high performance liquid chromatography (HPLC), ion exchangechromatography, Reverse Phase® chromatography and affinitychromatography (e.g. on SEPHAROSE™ (bead form gel prepared from agarose)Blue CL-6B or on carrier-bound monoclonal antibodies).

According to the invention, a tau protein or a fragment thereofcontaining the tau core fragment is incubated with a tau proteintogether with an agent suspected of being capable of modulating orinhibiting pathological tau-tau association. The extent of tau-taubinding which is correlated to the capacity of inhibition of the agentmay be detected by various methods:

In a preferred method a tau protein or a fragment thereof containing thetau core fragment is incubated with a tau derivative which is distinct,preferably immunologically distinct, from the first tau protein. In thiscase, binding of the tau derivative is detected for example via a poly-or monoclonal antibody or a derivative thereof. An example for this kindof detection is an assay method for the detection of tau-tau bindingcharacterised in that a truncated tau protein corresponding to the corefragment is incubated together with a test substance and either afull-length tau protein or a truncated tau protein fragment simulatingthe core PHF tau unit in the aqueous phase (FIGS. 8 and 10).

In this case, tau-tau-binding can be detected immunochemically in aconventional manner using an antibody which recognises the N-terminalsegment of the full length tau protein or, for example, an antibody suchas mAb 423 which recognises the core tau fragment truncated at Glu-391.Advantageously, the monoclonal antibody of the invention itself carriesa marker or a group for direct or indirect coupling with a marker asexemplified hereinafter. Also, a polyclonal antiserum can be used whichwas raised by injecting the corresponding tau antigen in an animal,preferably a rabbit, and recovering the anti-serum by immuno-affinitypurification in which the polyclonal antibody is passed over a column towhich the antigen is bound and eluting the polyclonal antibody in aconventional manner.

A particularly advantageous embodiment of the method of the inventioncomprises the use of an antibody directed against a human-specificsegment between Gly-16 and Gln-26 near the N-terminus of the tauprotein. The use of this kind of antibody makes it possible to measurebinding of full-length recombinant human tau to full-length tau isoformsderived from other animal species, for example rat, at various stages ofdevelopment. The binding of truncated tau can be detected by using anantibody such as mAb 423 to detect a truncated core tau fragmentterminating at Glu-391 binding to a similar fragment terminating atAla-390 not recognised by mAb 423. (FIG. 8)

The antibodies or fragments thereof may be used in any immunoassaysystem known in the art including, but not limited to:radioimmuno-assays, “sandwich”-assays, enzyme-linked immunosorbentassays (ELISA), fluorescent immuno-assays, protein A immunoassays, etc.

Particularly preferred is the following configuration for tau-taubinding assays (FIG. 10): A tau fragment, preferably a recombinant taufragment, corresponding to the truncated tau unit of the core PHF isbound to a solid phase, e.g. a conventional ELISA plate, in bufferconditions which have been shown not to favour tau-tau association. Thetruncated tau protein is preferably bound passively to the solid phase,since this has been found to expose the high affinity tau-tau bindingsite within the tandem repeat region. The solid phase is usuallypoly(vinyl-chloride), but may be other polymers such as cellulose,polyacrylamide, nylon, polystyrene or polypropylene. The solid supportsmay be in the form of tubes, beads, discs or micro plates, or any othersurfaces suitable for conducting an assay, and which on passive bindingof tau protein, exposes the high affinity tau capture site. Followingbinding, the solid phase-antibody complex is washed in preparation forthe test sample.

Surprisingly, appropriate buffer conditions for binding of the truncatedtau unit of the core PHF to a solid substrate without self-associationand without disturbance to the high affinity tau capture site within thetandem repeat region could be determined. An assay system wasestablished as shown in FIG. 8, in which the core tau unit truncated atAla-390 was first bound to the solid phase matrix. Next, a truncatedunit terminating at Glu-391 was incubated. Only the lafter could bedetected as mAb 423 immunoreactivity. FIG. 9 demonstrates thespecificity of the assay, in that mAb 423 immunoreactivity is seen onlyin the condition in which tau-tau binding is expected. An alkalinebuffer (sodium carbonate, tris, etc.), preferably pH 9-10, e.g. sodiumcarbonate buffer (50 mM, pH 9.6) was found to be associated withnegligible self association of core tau units (FIG. 9). Thereforeplating of the core tau unit for passive binding to solid phase matrixwas carried out in this buffer. If desired, a depolymerised tubulinpreparation or a preparation of microtubules in the same buffer can beplated for passive binding for determination of tau-tubulin binding.Suitable agents for blocking excess binding sites are milk extract,bovine serum albumin, gelatine, etc. After transfer of the solid phasebound core tau unit to physiological buffer conditions and incubationwith full-length tau in the standard binding assay format (FIG. 10), itwas possible to demonstrate extremely high affinity capture of normalfull-length tau protein. No binding of full-length tau was seen withoutprior plating of the core tau unit in the solid phase. When both specieswere present, binding was seen to depend on concentration of bothspecies. It was found that when either the solid-phase or aqueous phasespecies was saturating, the binding constant for the other species was8-25 nM, depending on the particular isoform of tau measured (FIG. 11).The buffer conditions for tau-tau binding should comprise suitable saltconcentrations and suitable pH values (FIGS. 12 and 13). The saltconcentrations for tau-tau binding should amount to preferably 50 to 400mM sodium chloride, more preferably 100 to 200 mM sodium chloride or acorresponding salt or salt mixture with a comparable ionic strength,e.g. PBS (137 mM sodium chloride, 1.47 mM potassium dihydrogenphosphate, 8.1 mM disodium hydrogen phosphate, 2.68 mM potassiumchloride). The pH range should comprise pH values of pH 4 to pH 10 andmore preferably pH 5 to pH 8. In order to saturate excess binding sitesand to avoid non specific binding the solid phase may be incubated witha blocking agent, e.g. milk extract, bovine serum albumin or preferablygelatine. After transfer of the passively bound core tau unit tophysiological buffer conditions, it was possible to demonstrateextremely high affinity capture of normal full-length tau protein(Kd=8-25 nM, depending on the particular tau species tested).

A liquid phase containing a tau protein capable of binding to the tauprotein of the solid phase is added together with the test substance tothe solid phase tau protein for a period of time sufficient to allowbinding. The bound tau complex is again washed in preparation foraddition of the antibody which selectively detects the secondarily boundtau species, but not the initial solid-phase species. The antibody islinked to a reporter molecule, the visible signal of which is used toindicate the binding of the second tau protein species.

Alternatively, detection of binding may be performed with a secondantibody capable of binding to a first unlabelled, tau specificantibody. In this case, the second antibody is linked to a reportermolecule.

By “reporter molecule”, as used in the present specification is meant amolecule which by its chemical nature, provides an analyticallydetectable signal which allows the detection of antigen-bound antibody.Detection must be at least relatively quantifiable, to allowdetermination of the amount of antigen in the sample, this may becalculated in absolute terms, or may be done in comparison with astandard (or series of standards) containing a known normal level ofantigen.

The most commonly used reporter molecules in this type of assay areeither enzymes or fluorophores. In the case of an enzyme immunoassay anenzyme is conjugated to the second antibody, often by means ofglutaraldehyde or periodate. As will be readily recognised, however, awide variety of different conjugation techniques exist, which are wellknown to the skilled artisan. Commonly used enzymes include horseradishperoxidase, glucose oxidase, β-galactosidase and alkaline phosphatase,among others.

The substrates to be used with the specific enzymes are generally chosenfor the production, upon hydrolysis by the corresponding enzyme, of adetectable colour change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase conjugates; for peroxidaseconjugates, 1,2-phenylenediamine or tetramethylbenzidine are commonlyused. It is also possible to employ fluorogenic substrates, which yielda fluorescent product rather than the chromogenic substrates notedabove. In all cases, the enzyme-labelled antibody is added to thecorresponding tau-tau protein complex and allowed to bind to thecomplex, then the excess reagent is washed away. A solution containingthe appropriate substrate, hydrogen peroxide, is then added to thetertiary complex of antibody-antigen-labelled complex. The substratereacts with the enzyme linked to the antibody, giving a qualitativevisual signal, which may be further quantitated, usuallyspectrophotometrically, to give an evaluation of the amount of antigenwhich is present in the serum sample.

Alternately, fluorescent compounds, such as fluorescein or rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic colour visually detectable with a light microscope.As in the enzyme immunoassay (EIA), the fluorescent-labelled antibody isallowed to bind to the first antibody-tau-peptide complex. After washingthe unbound reagent, the remaining ternary complex is then exposed tolight of the appropriate wavelength, and the fluorescence observedindicates the presence of the antigen.

In another preferred embodiment, the second tau protein species which isadded in liquid phase together with a test substance may be linked to areporter molecule as mentioned above. The second tau species may bedirectly modified (e.g. marked with a radioactive or enzymaticallydetectable label) or conjugated (e.g. to a fluorophore) in a domain ofthe molecule, for example the N-terminal segment, which is known not tobe involved in the high affinity tau-tau binding site, and therebyitself function both as the ligand in the tau-tau binding assay, and asthe reporter molecule.

A particular preferred embodiment of the present invention is describedin detail in Example 1.

The antibodies or fragments thereof used in the method of the presentinvention may be produced by conventional techniques, i.e. monoclonalantibodies which are selective to tau epitopes may be prepared by themethod of Köhler and Milstein. Suitable monoclonal antibodies to tauepitopes can be modified by known methods to provide Fab fragments or(Fab′)2 fragments, chimeric, humanised or single chain antibodyembodiments.

Examples for monoclonal antibodies being useful both to measure bindingaffinity in the tau-tau interaction, and to demonstrate theimmunochemical relationship between the binding demonstrated in vitroand that which occurs in the human brain are presented in the following:

Monoclonal antibodies recognising an N-terminal or C-terminal tauepitope permit measuring of binding between truncated and full lengthtau species. Especially useful are antibodies recognising human specificepitopes. A monoclonal antibody (designated AK 499) recognises a humanspecific epitope located in the region between Gly-16 and Gln-26 of tau,and thereby also permits measurement of binding between full-length tauspecies, provided one is derived from a non-human source (Lai (1995) Therole of abnormal phosphorylation of tau protein in the development ofneurofibrillary pathology in Alzheimer's disease. PhD Thesis, Universityof Cambridge). Antibody 342 recognises an non-species specific generictau epitope located between Ser-208 and Asn-265 (FIG. 21, SEQ ID NO: 4)which is partially occluded in the course of the tau-tau interaction(Lai, loc. cit.).

Other useful antibodies have already been described: antibody 423recognises tau C-terminally truncated at Glu-391 (Novak et al. (1993),loc. cit.). This truncation occurs naturally in the course of PHFassembly in Alzheimer's disease (Mena er al. (1995), (1996), loc. cit.;Novak et al. (1993), loc. cit.; Mena et al. (1991), loc. cit.). The sameC-terminal truncation can be demonstrated in vitro after binding offull-length tau to a truncated tau fragment terminating at Ala-390,which is not recognised by mAb 423) (Novak et al. (1993), loc. cit.),followed by digestion with the broad-spectrum protease, Pronase (FIG.16). In this configuration, the only possible source of mAb 423immunoreactivity is from digestion of bound full-length tau, and thiscan be shown to increase in a concentration-dependent manner withincreasing Pronase (FIG. 17A). This demonstrates that the molecularconformation of the tau-tau binding interaction generated in vitrocorresponds precisely to that which occurs in the brain, and hence thatselective inhibition of binding demonstrated in vitro can be generalisedto the human brain.

Antibody 7.51 recognises a generic tau epitope located in theantepenultimate repeat of tau (Novak et al. (1991) Proc. Natl. Acad.Sci. USA, 88, 5837-5841), which is occluded when tau is bound in aPHF-like immunochemical configuration but can be exposed after formicacid treatment (Harrington et al. (1990), (1991), loc. cit.; Wischik etal. (1995a), loc. cit.). Normal soluble tau, or tau bound tomicrotubules, can be detected by mAb 7.51 without formic acid treatment(Harrington et al. (1991), loc. cit.; Wischik et ab. (1995a), loc.cit.). Binding of full-length tau in the tau-tau binding assay isassociated with partial occlusion of the mAb 7.51 epitope.

In practicing the invention phenothiazines were identified whichproduced an inhibition of binding with a Ki of 98-108 nM (FIG. 19).Inhibition of 20% can be demonstrated at 1:1 molar ratio with respect totau, and further inhibition is approximately linear in the range up to10:1 molar ratio. These findings are consistent with the followingassumptions: tau-tau binding is determined by a finite number ofsaturable binding sites, and hence is specific; there is noco-operativity, i.e. that the binding of one molecule of tau does notinfluence the binding of a further molecule of tau at the site at whichinhibition occurs; binding is reversible, and is in a state of dynamicequilibrium in which binding is determined only by concentration andbinding affinity.

Given that the tandem repeat region of tau normally functions as thetubulin binding domain, and that the same region of the molecule alsocontains the high affinity tau capture site responsible for PHFassembly, it would only be possible to envisage a pharmaceuticalintervention to prevent pathological binding of tau if a more subtlemolecular difference could be demonstrated between the two types ofbinding, which would permit selective inhibition of pathological tau-tauinteraction, without inhibition of normal tau-tubulin binding, sincemany normal cellular processes, including particularly axonal transportof synaptic vesicles (Okabe and Hirokawa (1990) Nature, 343, 479-482),are dependent on the capacity of the cell the maintain tubulin in thepolymerised state. Prior experiments demonstrated immunochemicaldifferences (occlusion of the mAb 7.51 epitope in the tau-tau bindinginteraction, but no occlusion in the tau-tubulin binding interaction;Harrington et al. (1991), loc. cit.; Novak et al. (1991), loc. cit.) andmolecular differences (tau bound in a PHF-like configuration shows a14/16 amino acid residue phase-shift with respect to the normaltubulin-binding segment/linker segment organisation of the tubulinbinding domain which can be demonstrated by characteristic N- andC-terminal proteolytic cleavage sites; Novak, et al. (1993), loc. cit.,FIG. 3). Surprisingly, these differences could also provide a basis forpharmaceutical discrimination using small molecules withinwell-established pharmaceutical classes. In particular, the effects ofthe phenothiazines which were shown to inhibit pathological tau-tauassociation were tested for inhibition of normal tau-tubulin binding.Essentially no inhibition of binding could be demonstrated up to a molarratio of 1000:1 with respect to tau (FIG. 20). Nevertheless,hyper-phosphorylation of tau, which has been shown to inhibit the tautubulin-binding interaction, was also shown to produce comparableinhibition in this tau-tubulin binding assay (Lai, loc. cit.). Thus,compounds provided by the present invention which inhibit pathologicaltau-tau association do not inhibit normal binding of tau to tubulin.This represents the critical discovery of the present invention, sinceit demonstrates the technical feasibility of discovering compounds onthe basis of the screening system described herein which can distinguishpharmaceutically between the pathological binding of the tandem repeatregion in the PHF and the normal binding of the tandem repeat region inthe tau-tubulin interaction.

The only microtubule-associated protein identified so far within the PHFcore is tau protein. Nevertheless, PHFs assemble in the somatodendriticcompartment where the predominant microtubule-associated protein is MAP2(Matus, A. In Microtubules (Hyams and Lloyd, eds) pp 155-166, John Wileyand Sons, NY). MAP2 isoforms are almost identical to tau protein in thetandem repeat region, but differ substantially both in sequence andextent of the N-terminal domain (FIGS. 25 and 26, SEQ ID NO: 9 and 10).As shown in Example 3 aggregation in the tandem-repeat region is notselective for the specific tau core amino acid sequence, and theinhibitory activity of phenothiazine inhibitors such as thionine is notdependent on sequences unique to tau.

In addition, the present invention also relates to the corresponding invivo methods. These methods refer to the screening for agents thatmodulate or inhibit pathological tau-tau association characterised incontacting a cell line transfected either with tau protein or aderivative thereof containing the tau core fragment or with a vectorcapable of expressing a tau protein or a derivative thereof containingthe tau core fragment with an agent suspected of being capable ofmodulating or inhibiting tau-tau association followed by detection ofthe cell line viability and/or the cell line morphology.

Example 4 and 5 reveal that fibroblasts are fully viable when expressingtransgenic full-length tau protein and the cytoskeletal distribution oftransgenic full-length tau protein is not disturbed by culturing cellswith a potent tau-tau binding inhibitor. The phenothiazine thionine doesnot appear to have substantial intrinsic toxicity. But fibroblasts areeither not viable or show gross morphological abnormalities whenexpressing the transgenic core tau unit of the PHF. The frequency ofviable transfectants and the expression level for truncated tau areincreased in a dose-dependent manner by growing cells in thioninefollowing transfection. Viable transfectants expressing truncated tauare dependent on thionine, and revert to abnormal forms with lowviability upon its withdrawal.

These findings therefore substantiate in a non-neuronal cell system themajor findings of the present invention, namely: that high levels ofPHF-core tau within the cell are toxic; that this toxicity can bereversed by compounds which are selective inhibitors of the pathologicaltau-tau binding interaction; and that such compounds do not disrupt thenormal binding of tau to tubulin in vivo. These findings aregeneraliseable to other experimental models, including inducibletransfection systems and direct transfection of cells with truncated tauprotein.

Although the foregoing results support the use of tau-tau bindinginhibitors in reversing the toxicity of the truncated tau unit, it isdesirable to establish neuronal models of these processes. In general,neuroblastoma cell lines undergo complex cytoskeletal changes in thecourse of differentiation which depend on a balance between thedevelopment of the microtubule-network and a corresponding developmentof the neurofilamen t network. Higher molecular weightmicrotubule-associated proteins (MAP1A, MAP1B) are thought to providecross-bridges between these cytoskeletal systems (Schoenfield et al.(1989) J. Neurosci. 9, 1712-1730). Direct interference with themicrotubule-system with depolymerising agents (Wisniewski and Terry(1967) Lab. Invest. 17, 577-587) or aluminium (Langui et al. (1988)Brain Res. 438, 67-76) is known to result in intermediate filamentcollapse with formation of characteristic whorls in the cytoplasm(Wischik and Crowther (1986) Br. Med. Bull. 42, 51-56). A similaraggregation of the neurofilament cytoskeleton can be seen to occurspontaneously in neuroblastoma cell lines which fail to differentiate.The role of MAPs in the formation of these aggregates is not at presentunderstood. However, the formation, accentuation and inhibition of theseaggregates represent indirect markers of the capacity of microtubularcytoskeleton to associate with and transport the neurofilamentcytoskeleton into newly formed neurites.

Examples 6 and 7 reveal that phenothiazine inhibitors like thionine arenot toxic for neuronal cell lines at concentrations up to 2 μM andthionine does not interfere with incorporation of transgenic tau proteininto the endogenous microtubule network. These phenothiazines arerequired for production of viable neuronal cell lines following stabletransfection with a plasmid expressing truncated tau. Moreover,constitutive expression of truncated tau accentuates the formation ofpNFH aggregates, whereas the latter is inhibited by expression offull-length tau. The formation of cytoplasmic pNFH aggregates isinhibited by phenothiazines like thionine and incorporation of pNFHimmunoreactivity into neuronal processes is facilitated by thesecompounds.

These findings demonstrate that stable transfection of neuronal celllines with truncated tau is inherently toxic and, by destabilising themicrotubule system in surviving cells, results in the formation ofpresumptive neurofilarnent aggregates which fail to be transported intodeveloping neurites. These effects can be inhibited by a compoundselected for its capacity to block tau-tau aggregation in vitro, andthis action is presumably mediated by a permissive effect on expressionof endogenous tau or other MAPs required to stabilise microtubules.Phenothiazines like thionine also have the unexpected capacity to blockneurofilament aggregation in untransfected cells, either by facilitatingneuronal differentiation, or by directly inhibiting the formation ofneurofilament aggregates. In addition to their potential utility inprevention of tau aggregation in Alzheimer's disease, such compounds mayhave additional potential utility in the treatment of diseasescharacietised by pathological neurofilament aggregation, such as motorneuron disease and Lewy body disease. Transgenic mice which overexpressneurofilament subunits have been found to develop neurofilamentaggregates selectively in large motor neurones which undergodegeneration, leading to muscle wasting and weakness (Cote et al. (1993)Cell 73, 35-46; Xu et al. (1993) Cell 73, 23-33). Otherneurodegenerative disorders, Pick's disease and Progressive SupranuclearPalsy, show accumulation of pathological truncated tau aggregatesrespectively in Dentate is Gyrus and in stellate pyramidal cells of theneocortex. The compounds which have been described also have utility inthese neurodegenerative disorders.

Accordingly, the present invention especially relates to the abovemethod wherein said cell line preferably is a fibroblast or a neuronalcell line, more preferably a fibroblast 3T3, a PC-12 or a NIE-115 cellline. These cell lines are transfected preferably with a truncated tauprotein, containing at least the core tau unit. The expression of thetau protein may be under constitutive or under inducible control or thetau protein species may be directly transfected.

The present invention refers also to compounds which modulate or inhibittau-tau association as obtainable by a any method described above.

Based on the above results, the present invention provides also the useof phenothiazines of the formula

wherein:R₁, R₃, R₄, R₆, R₇ and R₉ are independently selected from hydrogen,halogen, hydroxy, carboxy, substituted or unsubstituted alkyl, haloalkylor alkoxy;R₂ and R₈ are independently selected from hydrogen or

R₅ is selected from hydrogen, hydroxy, carboxy, substituted orunsubstituted alkyl, haloalkyl, alkoxy or a single bond;R₁₀ and R₁₁ are independently selected from hydrogen, hydroxy, carboxy,substituted or unsubstituted alkyl, haloalkyl, alkoxy or a single bond;and pharmaceutically acceptable salts thereof in the manufacture of acomposition for the prophylaxis and treatment of pathological tau-tau orpathological neurofilarnent aggregation, and especially for theprophylaxis and treatment of Alzheimer's disease, motor neuron and Lewybody disease.

The term “alkyl” as used herein refers to straight or branched chaingroups, preferably having one to eight, more preferably one to six,carbon atoms. For example, “alkyl” may refer to methyl, ethyl, n-propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl,tert-pentyl, hexyl, isohexyl, and the like. Suitable substituents forthe substituted alkyl groups used in the invention include the mercapto,thioether, nitro, amino, aryloxy, halogen, hydroxyl, and carbonyl groupsas well as aryl, cycloalkyl and non-aryl heterocyclic groups.

The terms “alkoxy” refers to groups as defined herein above as alkylgroups, as the case may be, which also carry an oxygen atom interposedbetween them and the substrate residue to which they are attached.

The term “haloalkyl” represents a straight or branched alkyl chainhaving from one to four carbon atoms with 1, 2 or 3 halogen atomsattached to it. Typical haloalkyl groups include chloromethyl,2-bromethyl, 1-chloroisopropyl, 3-fluoropropyl, 2,3-dibrombutyl,3-chloroisobutyl, iodo-t-butyl, trifluoromethyl and the like.

The “halogen” represents fluoro, chloro, bromo or iodo.

Some compounds of the invention possess one or more asymmetricallysubstituted carbon atoms and therefore exist in racemic and opticallyactive forms.

The invention is intended to encompass the racemic forms of thecompounds as well as any of the optically active forms thereof.

The pharmaceutically acceptable acid addition salts are formed betweenbasic compounds of formula (I) and inorganic acids, e.g. hydrohalicacids such as hydrochloric acid and hydrobromic acid, sulphuric acid,nitric acid, phosphoric acid etc., or organic acid, eg. acetic acid,citric acid, maleic acid, fumaric acid, tartaric acid, methanesulphonicacid, p-toluenesulphonic acid etc.

In a particular preferred embodiment the present invention provides theabove phenothiazine wherein

R₁, R₃, R₄, R₆, R₇ and R₉ are independently selected from -hydrogen,

—CH₃, —C₂H₅, or —C₃H₇;

R₂ and R₈ are independently selected from

wherein R₁₀ and R₁₁ are independently selected from a single bond,hydrogen, —CH₃, —C₂H₅, or —C₃H₇;R₅ is a single bond, -hydrogen, —CH₃, —C₂H₅, or —C₃H₇ andpharmaceutically acceptable salts thereof.Especially preferred are following phenothiazines

Compounds useful for the blocking of pathological tau-tau association,preferably phenothiazines (FIGS. 23 and 24), are characterised by abinding coefficient of less than 0.4, and lack of inhibition in thetau-tubulin binding assay, preferably up to a molar ratio of 1000:1 withrespect to the molar concentration of tau.

The phenothiazines of the present invention are known in the art and maybe manufactured by the processes referred to in standard texts (e.g.Merck Manual, Houben-Weyl, Beilstein III/IV 27, 1214 ff, J. Heterocycl.Chem 21, 613 (1984), etc.).

The compounds of the above formula, their pharmaceutically acceptablesalts, or other compounds found to have the properties defined in theassays provided, could be used as medicam.ents after further testing fortoxicity (e.g. in the form of pharmaceutical preparations). The priorpharmaceutical use of methylene blue in a wide range of medicalindications has been described, including treatment ofmethaernoglobineamia and the prophylaxis of manic depressive psychosis(Naylor (1986) Biol. Psychiatry 21, 915-920), and CNS penetrationfollowing systemic administration has been described (Müller (1992) ActaAnat., 144, 39-44). The production of Azure A and B occur as normalmetabolic degradation products of methylene blue (Disanto and Wagner(1972a) J. Pharm. Sci. 61, 598-602; Disanto and Wagner (1972b) J. Pharm,Sci. 61, 1086-1094). The administration of pharmaceuticals can beeffected parentally such as orally, in the form of tablets, coatedtablets, dragees, hard and soft gelatine capsules, solutions, emulsionsor suspensions), nasally (e.g. in the form of nasal sprays) or rectally(e.g. in the form of suppositories). However, the administration canalso be effected parentally such as intramuscularly or intravenously(e.g. in the form of injection solutions).

For the manufacture of tablets, coated tablets, dragees and hardgelatine capsules the compounds of formula I and their pharmaceuticallyacceptable acid addition salts can be processed with pharmaceuticallyinert, inorganic or organic excipients. Lactose, maize starch orderivatives thereof, talc, stearic acid or its salts etc. can be used,for example, as such excipients for tablets, dragees and hard gelatinecapsules.

Suitable excipients for soft gelatine capsules are, for example,vegetable oils, waxes, fats, semi-solid and liquid polyois etc. Suitableexcipients for the manufacture of solutions and syrups are, for example,water, polyols, saccarose, invert sugar, glucose etc.

Suitable excipients for injection solutions are, for example, water,alcohols, polyols, glycerol, vegetable oils etc.

Suitable excipients for suppositories are, for example, natural orhardened oils, waxes, fats, semi-liquid or liquid polyols etc.

Moreover, the pharmaceutical preparations can contain preserving agents,solubilizers, viscosity-increasing substances, stabilising agents,wetting agents, emulsifying agents, sweetening agents, colouring agents,flavouring agents, salts for varying the osmotic pressure, buffers,coating agents or antioxidants. They can also contain still othertherapeutically valuable substances.

In accordance with the invention the compounds of the above formula andtheir pharmaceutically acceptable salts can be used in the treatment orprophylaxis of Alzheimer's disease, particularly for the blocking,modulating and inhibiting of pathological tau-tau association. Thedosage can vary within wide limits and will, of course, be fitted to theindividual requirements in each particular case. In general, in the caseof oral administration there should suffice a daily dosage of about 50mg to about 700 mg, preferably about 150 mg to about 300 mg, divided inpreferably 1-3 unit doses, which can, for example, be of the sameamount. It will, however, be appreciated that the upper limit givenabove can be exceeded when this is found to be indicated.

The invention can be understood better when they are read in conjunctionwith the accompanying figures:

FIG. 1: Representation of tau protein binding to microtubules (modifiedafter Butner and Kirschner, loc. cit.).

FIG. 2: Schematic representation of tau protein isoforms, withcorresponding amino acid and cDNA sequences shown in FIG. 21. TheN-terminal domain of 252 residues contains either one or two insertsamounting to a further 58 residues (“1”, “2”), followed by a tandemrepeat region of 93-125 residues containing 3 or 4 tandem repeats, and aC-terminal tail of 64 residues. The tau fragments isolated from enrichedprotease-resistant PHF-core preparations are denoted “F5.5”, and consistof a mixture of species derived from both 3- and 4-repeat isoforms, butencompassing 93-95 residues, the equivalent of 3-repeats, phase shiftedby 14 16 residues with respect to the normal organisation of the tandemof tandem repeat region. All F5.5 species and normal tau are recognisedby mAb 751, but mAb 423 recognises only those F5.5 fragments terminatingat Gln 391. The positions of epitopes for mAb's 499, AT8 and 342 arealso shown.

FIG. 3: N-terminal sequence analysis of the 12 kDa F5.5 fragmentreleased from core PHF preparations revealed the presence of 6 distinctpeptides which can be grouped into 3 pairs derived from 3-repeat (A:repeats 1-3; SEQ ID NO: 1) or 4-repeat (B: repeats 1-3; SEQ ID NO: 2, orC: repeats 2-4; SEQ ID NO: 3) isoforms (Jakes et al., loc. cit.). mAb423immunoreactivity serves to define a C-terminal boundary at Glu-391(shown by arrow, Novak et al. (1993), loc. cit.). The N- and C-terminalboundaries indicated by vertical arrows in FIGS. 3A to 3C thus serve todefine a phasing of the tandem repeat region within the PHF core whichis shifted 14-16 residues with respect to the sequence homology repeats.This minimal protease resistant core PHF tau unit is 93/95 residues longwhich is precisely equivalent to 3 repeats. The boundaries of this unitare also out of phase with respect to the tubulin binding domainsproposed by Butner and Kirschner (loc. cit.), which are shownunderlined.

FIG. 4: Total tau protein content in controls and Alzheimer's disease.Normal soluble tau (white) is the predominant form found in controls,whereas in Alzheimers disease, the predominant form of tau ispolymerised into PHFs (black).

FIG. 5: Changes in soluble tau, phosphorylated tau, and tangle countduring early stages of Alzheimer's disease (Lai et al. (1995), loc.cit.). The accumulation of PHF-bound tau is shown on the horizontalaxis. This is accompanied by a relative loss in normal soluble tau. Thefirst appearance of phosphorylated tau is closely linked to the firstappearance of tangles. However, both of these appear only after asubstantial redistribution of tau from soluble to polymerised phases hasalready occurred.

FIGS. 6A and 6B: Calculated rates of transfer of new tau synthesis intothe soluble tau pool (FIG. 6A), and of soluble tau into the PHF-boundpool (FIG. 6B) at early stages of Alzheimer's disease (Lai et al.(1995), loc. cit.). As the soluble tau level drops below 580 pmol/g,progressively more new tau synthesis is required to keep pace with therate of PHF production, and this appears to be regulated in a negativefeedback manner with respect to the ambient level of soluble tau (FIG.6A). The rate of transfer of soluble tau into PHFs is geometric withrespect to the ambient level of PHF-tau (FIG. 6B).

FIG. 7: Hypothetical scenario for transformation of tau protein intoPHFs in Alzheimer's disease. Once tau has been immobilised andtruncated, a high affinity pathological tau capture site is exposed.When a further molecule of tau is captured, only partial proteolyticdegradation is possible, since the region of high affinity tau-tauassociation is protected from proteolysis, leaving a further highaffinity tau capture site available for the capture of a further taumolecule. The redistribution of the tau protein pool from soluble totruncated PHF-bound phases is autocatalytic, mediated by repetitive highaffinity tau capture and partial proteolysis.

FIG. 8: Tau binding assay configuration in which binding of twotruncated units is measured. The species terminating at Ala-390 (“a”) isfirst coated on the ELISA plate (in sodium carbonate buffer: 50 mM, pH9.6). Next, a second truncated tau species terminating at Glu-391 (“e”)is incubated in various buffer conditions shown in FIG. 9. Only thespecies “e” is recognised by mAb 423, and hence mAb 423 immunoreactivitymeasures only that tau which is bound during the second incubation.

FIG. 9: Binding of species “e” (0 or 20 μg/ml) to “a” (0 or 10 μg/ml) inphosphate buffered normal saline (“normal”), distilled water (“water”)and sodium carbonate buffer (“carbonate”, 50 mM, pH 9.6). The verticalaxis shows mAb 423 immunoreactivity. No immunoreactivity is detectedwhen species “a” is coated alone, because mAb 423 does not recognise“a”. No immunoreactivity is detected when “e” is incubated without priorplating of “a”. This is because the blocking conditions used preventnon-specific binding of “e” to the ELISA plate. Immunoreactivity is onlyseen in the condition in which “a” and “e” are both present,demonstrating the specific detection only of “e” which is has been boundto “a”. No binding is seen when “e” is added in sodium carbonate buffer.Therefore, this condition represents the optimal one for initial platingof “a”, since self-aggregation is minimised in this condition.

FIG. 10: Standard configuration for measurement of binding offull-length tau (“t”) to the truncated core tau unit previously boundpassively to the solid phase (“a”). A recombinant tau fragment (“a”)corresponding to the truncated tau unit of the core PHF is plated atvarying concentrations on an ELESA plate in conditions which have beenshown not to favour tau-tau association (FIG. 9). After blocking, fulllength recombinant tau (“t”) is plated in conditions which permitselective detection of tau-tau binding. Binding is detected by anappropriate antibody, which recognises an epitope located near theN-terminus of full-length tau. This antibody does not recognise “a”.

FIG. 11: Determination of Kd for binding of full-length tau (“T40”) tothe truncated core tau unit terminating at Ala-390 (“a”), using mAb 499to measure bound full-length human tau. The horizontal axis on the uppergraph shows the concentration of T40 used and the vertical axis showsmAb 499 immunoreactivity. Each binding curve is obtained at a platingconcentration of “a” which is shown. Without “a”, there is no binding,confirming the absence of non-specific binding of T40 in the assayconditions used. Binding depends both on the concentration of T40 andthe concentration of “a”. The lower figure shows the calculated Kdcorresponding to each plating concentration of “a”. As the concentrationof “a” becomes large, saturating conditions are approachedassymptotically, and this represents the saturation Kd for binding ofT40 to the truncated core tau unit, in this experiment determined as22.8 nM.

FIG. 12: Using the standard assay format shown in FIG. 10, with species“a” coated at 10 μg/ml and T40 added at the concentrations shown (range0-50 μg/ml), binding was measured at constant pH (pH 7.4), while varyingthe sodium chloride concentration. A plateau is observed in the vicinityof the physiological salt concentration of 137 mM. Binding is reduced atmoderately low and high salt concentrations, although binding becomesmore favourable at very low salt concentration.

FIG. 13: Similar experiment to that shown in FIG. 12, keeping the sodiumchloride concentration constant at 137 mM, but varying the pH in therange 0-10, with binding in physiological phosphate-buffered normalsaline (“PBS”, pH 7.4) shown for comparison. Binding is reduced atextremes of pH. Binding shown detected by mAb's 499 and 342.

FIG. 14: Typical sets of binding curves using the truncated core tauunit “a” in the solid phase, and incubating full length tau which has(“T4OP”) or has not (“T40”) been phosphorylated in vitro using themethod of Biernat et al. (1992) EMBO J. 11, 1593-1597). The Kd wasreduced by phosphorylation in this experiment by 10-fold, althoughvarying the state of phosphorylation in the aqueous and solid phasessystematically, the overall effect of phosphorylation can be shown to beon aveage 20-fold inhibition of binding. Although a fetal state ofphosphorylation has been proposed by some as important for determiningpathological tau-tau binding, fetal rat tau (“POTr”) when introduced inthe aqueous phase is shown here to be incapable of pathological bindingto the core tau unit.

FIGS. 15A and 15B: By contrast with FIG. 14, after fetal tau has beenbound passively in the solid phase, it is able to bind full-lengthunphosphorylated tau. A typical set of binding curves is shown in FIG.15A, varying the concentration of full-length tau (“T40”) and fetal tau(“PO Tau”) in the concentration ranges shown. The derived assymptotic Kdis shown in FIG. 15B. As with binding of the full-length tau to thetruncated core tau unit, binding of full-length tau to immobilised fetaltau has the same Kd of ˜20 nM. Thus fetal tau, which does not bind totau when it is present in the aqueous phase (FIG. 14), is converted intoa tau-binding species simply by passive binding to the solid phase. Thuspassive binding of tau to a solid matrix exposes the high affinity taucapture site.

FIG. 16: Comparison of Kd values in the tau-tau binding assay using thespecies shown in the aqueous or solid phases. Phosphorylation of fulllength recombinant tau used in the aqueous phase inhibits binding by afactor of 10-fold, and foetal/newborn tau from rat does not bind, asshown in FIG. 14. When newborn tau is used in the solid phase, T40 bindswith the same affinity as to the truncated core PHF unit.Phosphorylation of T40 in the aqueous phase produces 30-fold inhibitionof binding. Hyperphosphorylation of newborn tau in the solid phaseinhibits binding to a comparable extent, and hyperphosphorylation inboth phases produces 50-fold inhibition of binding. Therefore, contraryto the phosphorylation hypothesis, phosphorylation inhibits thepathological self-aggregation of tau protein in all configurations ofthe present assay.

FIGS. 17A, 17B and 17C: Proteolytic digestion of aggregated full-lengthtau protein. (FIG. 17A) Full-length tau (20 μg/ml) was bound to dGA (20μg/ml) in PBS, washed, and incubated for 5 min with Pronase in water atthe concentrations indicated. Immunoreactivity was measured with mAb's342 (Δ), 499 (∘) and 423 (●). (FIG. 17B) Full length tau (10 μg/ml)which had self-aggregated in the solid phase in the absence of dGA wasdigested similarly, and immunoreacticity was measured with mAb's 342 (Δ)and 423 (●). In both cases, protease concentration-dependent loss ofimmunoreactivity with both mAb's 499 and/or 342 occurred with theacquisition of mAb 423 immunoreactivity. (FIG. 17C) The results from(FIG. 17A) are depicted schematically. Truncated dGA, initially coatedon the hatched solid phase, binds full-length tau with high affinitythrough interaction via the repeat region. Both species lack the mAb 423epitope prior to digestion. Proteolytic digestion of the complex (dottedlines) removes the N-terminal portion of the full-length tau moleculewith loss of the mAb 499 and 342 epitopes located as shown. Acquisitionof immunoreacticity with mAb 423 indicates truncation of full-length tauat Glu-391. The precise N-terminal extent of the proteolytically stablecomplex is unknown, but excludes the mAb 342 epitope immediatelyadjacent to the repeat region, and includes the tau-binding domain.

FIGS. 18A and 18B: Accumulation of truncated tau by repetitive taucapture. Beginning with the truncated tau fragment (dGA, 20 μg/ml) inthe solid phase, full-length recombinant human tau (20 μg/ml) was bound,digested with Pronase (1 ng/ml) for 5 min. washed, and the preparationwas again incubated with further full-length tau (20 μg/ml) and againdigested. This binding/digestion cycle was repeated four times; mAb 499immunoreactivity was measured before and after, and mAb 423 measuredonly after, each Pronase digestion step. (FIG. 18A) Pronase digestion ofthe complex was associated with incremental accumulation of tau proteintruncated at Glu-391 in the solid phase following each digestion cycle.(FIG. 18B) Binding of full-length tau was detected by the appearance ofimmunoreactivity for the N-terminus of tau (mAb 499), which was entirelyabolished by Pronase digestion. In the subsequent incubation cycle, thebinding capacity was increased for full-length tau incubated at aconstant concentration in the aqueous phase. The incremental mAb 499immunoreactivity cannot be explained by residual immunoreactivity leftfrom the preceding cycle. Thus the proteolytically stable complex leftafter Pronase digestion retains the capacity to bind further tau, andthis binding capacity increases as truncated tau accumulates in thesolid phase.

FIGS. 19A and 19B: Relative tau-tau binding (vertical axis) in thepresence of increasing concentrations of prototype inhibitoryphenothiazines (horizontal axis). This inhibition can be expressed interms of a standard competitive inhibition model, with calculated Ki of98-108 nM. The correlation coefficients for these approximations are0.99, and are highly significant statistically, as shown. In FIG. 19A,the abscissa, represented as [A], refers to Azure A concentration. InFIG. 19B, the abscissa, represented as [B], refers to thionineconcentration.

FIG. 20: Selective inhibition of tau-tau-binding by thionine. Truncatedtau protein was used at 489 nM in both aqueous and solid phases of theassay as in FIG. 8 (filled circles). In the tau-tubulin assay,depolymerised tubulin was coated at 200 nM (open circles), and tau wasincubated at 400 nM. Binding data could be described mathematically by astandaid model which assumes competitive inhibition at the high affinitytau capture site. The Ki values were calculated using the Kd valuesobtained from the corresponding binding studies using full-length tau.Data points represent means of quadruplicate measurements.

FIGS. 21A and 21B: Nucleotide and predicted amino acid sequences of ahuman tau protein isoform (SEQ ID NO: 4). The sequence, deduced fromcDNA clone htau40, differs from the previously determined three-repeatform (Goedert et al. (1988), loc. cit.) by an extra 58 amino acidsinserted in the amino-terminal region (underlined) an by the previouslydescribed (Goedert et al. (1989), EMBO J. 8, 393-399) extra repeat of 31amino acids (underlined). Nucleotides are numbered in the 5′-3′direction. The cDNA clone htau40 (Goedert et al. (1989b), Neuron 3,519-526) contains the above sequence inserted into an Ndel site (5′-end)and an EcoR1 site 3′ to the termination to the codon (***).

FIG. 22: Amino acid and cDNA sequence of PHF-core tau unit (SEQ ID NO:6; Novak et al. (1993), loc. cit.), and primers (SEQ ID NO: 7 and 8)used in construction of the. preferred core tau unit.

FIG. 23: Ranking of compounds by inhibition of tau-tau interaction.Ranking is based on the standardised binding relative to that seen inthe absence of compound taken as the mean observed at 1 and 10 μg/ml. Inthis ranking, “1” represents binding equivalent to that observed in theabsence of compound, whereas “0.2” indicates that binding was reduced toa mean of 20% at test compound concentrations 1 and 10 μg/ml. Thus thelower the number the more effective the compound at inhibiting thebinding of e and a. As can be seen, the first five phenothiazines havestandardised binding coefficients less the 0.4. That is, the bindingseen in the range 1-10 μg/ml is less than 40% of that seen in theabsence of compound.

FIGS. 24A-24D: Chemical structures of the compounds tested with valuesfor standardised binding according to FIG. 23.

FIG. 25: Schematic representation of tau, MAP2 (adult form), MAP2C(juvenile form) and high molecular weight tau (found in the peripheralnervous system and neuroblastoma cell lines). These proteins sharesimilar microtubule-binding domains, but differ substantially insequence and extent of the N-terminal projection domain. The juvenileforms of tau and MAP2 have only 3 of the tandem repeats. A 4-repeat formof MAP2 also exists.

FIG. 26: Sequence differences in the tandem repeat region of human tau(upper line; SEQ ID NO: 9) and mouse MAP2 (lower line; SEQ ID NO: 10).Vertical arrows show the limits of the truncated PHF-core fragmentterminating at Glu-391, and the tubulin-binding segments are shownunderlined.

FIG. 27: The pIF2 expression vector is an SV40-based eukaryoticexpression vector (pSV2neo; Sambrook et al. (1989), loc. cit.; SEQ IDNO: 11 and 12 modified to contain β-globin promotor driving theexpression of foreign DNA (M. N. Neuberger). It has a neomycinresistance marker for Geneticin selection.

FIG. 28: Mouse fibroblast 3T3 cells transfected with PIF2::T40,expressing full-length human tau protein (T40), imrnunolabelled by mAb7.51 (upper figure) and mAb 499 (lower figure). Cells form long slenderprocesses, and tau immunoreactivity is also seen to have a cytoskeletaldistribution in the perikaryon.

FIG. 29: Mouse fibroblast 3T3 cells transfected with PIF::dGAE,expressing the truncated PHF-core tau fragment terminating at Glu-391,immunolabelled with mAb 7.51. Early cell line transfected and grownwithout thionine. Cells are grossly abnormal, multinucleate, vacuolated,containing aggregates of tau protein in the cytoplasm.

FIG. 30: Lipofectin/tau protein transfers into 3T3 cells transfectedwith PIF2::T40. Relative cell survival (normalised to cell counts afterLipofectin treatment without protein) is shown for approximatelyequimolar concentrations of full-length (T40, 220 nM) and truncated tau(dGAE, 300 nM), without (unshaded) or with shaded) thionine at 1 μM.Truncated tau is more toxic than full-length tau (p=0.02), despite thefact that at equimolar concentrations, the total protein load is 5×greater in the case of full-length tau.

FIGS. 31A and 31B: (FIG. 31A) Reversal of truncated tau toxicity: Thetoxicity of truncated tau transferred via lipofectin into 3T3 cellsexpressing full-length tau is concentration dependent. Thionine(full-line) significantly reversed toxicity seen in the absence ofthionine (broken line) at all three concentrations of truncated tau.(FIG. 31B) Similar experiment in which full-length tau was transferredvia lipofectin into 3T3 cells expressing full-length. Both toxicity andthionine effects were much less apparent.

The following Examples are intended to illustrate details of theinvention, without thereby limiting it in any manner.

EXAMPLES Example 1 Tau-Tau-Binding Assay

The assay is carried out in a 96-well PVC microtitre plate, withsolutions added and readings taken with respect to individual wells:

a) A 50 μl solution of purified truncated tau peptide at varyingconcentrations ranging 0-50 μg/ml (0,1,5,10,50 μg/ml) in 50 mM sodiumcarbonate buffer (pH 9.6) is added to each well and incubated 1 hr at37° C.

b) The microtitre plate wells are washed 3× with water with or without0.05% TWEEN™ (polyoxyethylene derivatives of sorbitan esters).

c) A 200 μl solution of 2% milk extract (“Marvel”) made up inphosphate-buffered normal saline (“PBS”, 137 mM sodium chloride, 1.47 mMpotassium dihydrogen phosphate, 8.1 mM disodium hydrogen phosphate, 2.68mM potassium chloride) is added to each well and incubated for 1 hr at37° C.

d) The plate is washed as in b).

e) A 50 μl solution of full-length recombinant tau (T40) in the samerange of concentrations as in a) above in 1% gelatine, 0.05% TWEEN™(polyoxyethylene derivatives of sorbitan esters) in PBS is added to eachwell, and incubated for 1 hr at 37° C.

f) The plate is washed as in b).

g) A 50 μl solution of monoclonal antibody 499 is added at ½ dilution ofthe tissue culture supernatant with 2% milk extract (“Marvel”) in PBS isadded to each well and incubated for 1 hr at 37° C.

h) The plate is washed as in b).

i) A 50 μl solution of second antibody (blofting grade affinity purifiedgoat anti-mouse IgG (H+L) conjugated with horseradish peroxidase—Bioradcatalogue number 170-6516) at 1/1000 dilution in PBS with 0.05% TWEEN™(polyoxyethylene derivatives of sorbitan esters) is added to each welland incubated for 1 hr at 37° C.

j) The plate is washed 3× with a 0.05% solution of TWEEN™(polyoxyethylene derivatives of sorbitan esters) in water, followed by asingle wash with water.

k) Preparation of colour development solution is as follows. Dissolve10-15 mg of 3,3′,5,5′-tetramethylbenzidine (TMB; BCL catalogue number784 974) in dimethylsuphoxide to a final concentration of 10 mg/ml (TMBsolution). Add 10 ml sodium acetate stock (0.5 M, pH 5.0) to 90 ml ofwater. While swirling, slowly add 1 ml TMB solution, followed by 10 μlhydrogen peroxide.

l) A 50 μl solution of TMB solution is added to each well to develop theperoxidase colour reaction, the rate of development of which is readover 2 min. at 650 nm, in a Molecular Devices Microplate reader usingKinetic L1 Softmax software package.

Example 2 Preparation of Recombinant Tau Fragments

Tau cDNA was generated using standard protocols (Sambrook et al., loc.cit.) from mRNA isolated from brain tissue of an Alzheimer patient whosetissue was obtained 3 h after death. The cDNA library was screencd withsynthetic 17-mer oligonucleotide probes derived from the sequence frompart of a PHF core protein (Goedert et al. (1988), loc. cit.). FullLength cDNA. clones were subcloned into the EcoRI site of M13mp18 andsite-directed mutagenesis used to introduce a Ndel site in the contextof the initiator codon. Following cleavage with Ndel and EcoRI, theresulting CDNA fragments were subcloned downstream of the T7 RNApolymerase promotor into Ndel/EcoRI-cut expression plasmid pRK172(McLeod et al. (1987) EMBO J. 6, 729-736). pRK172 is a derivative ofpBR322 that is propagated at very high copy number in E. coli due toremoval of the pBR322 copy number control region. The plasmid carries anampicillin resistance gene for selection of recombinant clones.

Constructs coding for truncated forms of tau were prepared from mRNA asdescribed in Novak et al. (1993, loc. cit.). The mRNA was used as atemplate for polymerase chain reaction (PCR) using specificoligonucleotide primers. The sense primer contained an Ndel site and theanti-sense, an EcoRI site. PCR fragments were subcloned into pRK172 asdescribed above. The primers used fOr construction of dGAE are given inFIG. 22. The authenticity of all DNA fragments used for expression wasconfirmed by full length sequencing of both strands.

Details for the construction of htau40 (“T40”) cDNA are described in(Goedert et al. (1989), loc. cit.). This sequence is the largest form oftau found in the CNS and encodes tau protein that contains both the 2N-terminal inserts of 29 amino acids each and an extra 31 amino acidrepeat in the tubulin-binding domain. The DNA sequence and its predictedamino acid sequence are shown in FIG. 21 (SEQ ID NO: 4).

Recombinant plasmids were used to transform E. coli BL21 (DE3) a strainused for prokaryotic expression which carries a chromosomal copy of thebacteriophage T7 RNA polymerase gene under control of the lacUV5promotor (Studier and Moffat (1986), J. Mol. Biol. 189, 113-130).Exponentially growing cultures were induced with IPTG (iso-propylthiogalactoside) for 3 h.

Large-scale purification (1 liter bacterial culture) of tau fragmentswas carried out as described by (Goedert and Jakes (1990, EMBO J., 9,4225-4230), with minor modifications. Cells were disrupted by rapidfreezing of the cell pellet in liquid nitrogen. The pellets were thensuspended in buffer containing 50 mM PIPES, 1 mM dithiothreitol (DTT)(pH 6.8). The thermostable proteins in the supernatant were dialysedagainst PIPES/DTT, then applied to a column containing phosphocelluloseequilibrated in the same buffer. Tau protein was eluted with a gradientof NaCl (0-0.5M) in the above buffer. Fractions were analysed bySDS-PAGE and both Coomassie staining and immunoblotting. Those fractionscontaining tau were pooled, dialysed against 25 mM MES, 1 mM DTT (pH6.25) and stored at −20° C. at approximately 5 mg/ml. Proteinconcentrations were measured by the Lowry method (Harrington (1990),loc. cit.).

Example 3 Binding of Foetal MAP2C to Truncated and Full Length Tau

One possible explanation for the lack of MAP2 in PHFs might be that MAP2in PHFs might be MAP2 is unable to bind to the core tau unit of the PHFbecause of sequence differences in the repeat regions. This was examinedexperimentally using the standard binding assay in two configurations:truncated tau in the solid phase with foetal MAP2C in the aqueous phase,and MAP2C in the solid phase with full-length tau in the aqueous phase.Binding could be demonstrated in both configurations, ant thionineblocked the tau/MAP2 binding interaction. Thus, aggregation in thetandem-repeat region is not selective for tau, and the inhibitoryactivity of phenothiazine inhibitors such as thionine is not dependenton sequences unique to tau. The reason why MAP2 is not found in PHFs isat present unknown, but factors may include the contribution of thelarge N-terminal domain found in the adult form of MAP2, compartmentdifferences within the cell, or other differences in processing of theMAP2 molecules.

Example 4 Transfection of Mouse 3T3 Cells with Human Tau Protein

Mouse fibroblast 3T3 cells were transfected with a eukaryotic expressionvector (pIF2) containing full-length and truncated forms of tau proteinunder constitutive control by a β-globin promotor. This vector containsa neomycin resistance gene as a selectable marker (pSV2neo; Sambrook etal. (1989), loc. cit.; modified by M. N. Neuberger). Cells were culturedin defined minimal essential mixtures (DMEM) containing antimicrobialagents and 10% foetal calf serum at 37° C. in an atmosphere of 5% CO₂.They were transfected with plasmid DNA either using a standard calciumphosphate protocol or by lipofection (according to manufacturersprotocol; Gibco BRL). Cells which had integrated the plasmid DNA wereselected by viability in medium containing Geneticin (0.5 mg/ml;Southern and Berg (1982), J. Mol. Appl. Genet. 1, 327).

Stably transfected 3T3 fibroblast expressing full-length tau proteinwere readily produced. Expression could be demonstrated histologicallyusing generic (mAB 7.51) and human-specific (mAB 499) anti-tauantibodies (FIG. 28), and by immunoblot of cell extracts (not shown).Two viable cell lines were produced when the transfection was carriedout using the same vector carrying the truncated core tau unit.Truncated tau could be demonstrated within these cells histologically,but the morphology of these cells was grossly abnormal compared to thoseexpressing full-length tau (FIG. 29). Abnormalities included failure ofprocess development, formation of large rounded cells, cytoplasmicaggregation of tau and vacuolation of the cytoplasm. However, thesecells proved unstable, and readily reverted to forms failing to expresstruncated tau protein despite the continued presence of Geneticin. Thetoxicity of truncated tau might be explained either by the accumulationof toxic tau-tau aggregates in the cell or by the binding of truncatedtau to endogenous mouse MAPs essential for the cell.

Example 5 Growing of Tau-transfected Cells in the Presence ofPhenothiazine Inhibitors

The toxicity of the truncated core tau unit might be reversible in partif the prototype phenothiazine inhibitors could be used to blockself-aggregation in vivo. This would be feasible only if the compoundswere not intrinsically toxic at concentrations needed to block tau-taubinding. The inhibitors with the lowest toxicity in 3T3 cells werethionine and acriflavin, and cells could survive prolonged exposure tothese compounds at concentrations substantially in excess of the Kivalues (100 nM) for inhibition of tau-tau binding in vitro. In practice,3T3 cells could be grown several month in the presence of 2 μM thionine.

The influence of thionine on the tau-tubulin binding interaction wasexamined in vivo by culturing 3T3 fibroblast transfected withfull-length tau protein in the presence of thionine at a range ofconcentrations. Disruption of normal cytoskeletal distribution of tauimmunoreactivity was seen at concentrations in the range 4-8 μM,comparable with the known Ki. for inhibition of the tau-tubulin bindinginteraction in vitro (8 μM), but no effect was seen over theconcentration range at which transfected 3T3 cells were routinelycultured (0.5-2 μM). These findings demonstrate the feasibility ofculturing transfected cell lines in the presence of prototypic inhibitorwithout detriment either to cell viability or to the normal cytoskeletaldistribution of transgenic full-length tau protein.

Growing transfected cells in the presence of inhibitors of tau-taubinding was found to increase the viability of cells transfected withtruncated tau in a dose-dependent manner. The number of viable celllines transfected with truncated tau increased when the cells were grownin the presence of higher concentrations of thionine. Furthermore, thestrength of expression of truncated tau, measured byimmunohistochemistry on a semiquantitative scale, was found to increaseas a function of the thionine concentration used following transfection.

The morphology of 3T3 cells and the distribution of truncated tauprotein were much less abnormal when transfected cell lines wereproduced in the presence of thionine. Truncated tau protein appeared tofollow the distribution of the endogenous microtubule network, but thetau staining had a more broken character than seen with full length tau.Cells expressing high levels of truncated tau were found to formaggregates with gross disruption of the cell cytoplasm when thionine wasremoved. This was similar to the initial findings for cells transfectedin the absence of thionine.

Example 6 Untransfected Neuronal Cell Lines

Neuronal cell lines (N2A, NIE-115) were cultured in DMEM containing 2%or 10% foetal calf serum and 5% horse serum on tissue culture platescoated with collagen. These were all grown at 37° C. in an atmospherecontaining 5% CO₂. Initial immunohistochemical studies of neuronal celllines prior to transfection led to the identification of cytoplasmicaggregates immunoreactive with mAb 423 forming in the cytoplasm ofundifferentiated neuroblastoma cells (N2A cells) and in PC-12 cellsafter brief treatment with dibutyryl-cAMP (db-cAMP, known todifferentiate neuroblastoma cells in tissue culture). These structureswere shown to be immunoreactive with an antibody recognisingneurofilament protein (NFH; SMI-31, Sternberger et al. (1985) PNAS 82,4274-4276) and more sparesly immunoreactive with an antibody recognisingMAP1A, which is known to bind neurofilaments. In the course ofdifferentiation, this endogenous mAb 423 immunoreactivity was seen toshift from the cytoplasm to neurites. Immunoprecipitation of mAb423immunoreactivity from these cells led to the identification of a specieswith gel mobility of 230 kDa which was recognised by SMI-31. Theseresults suggest that the structures recognised by mAb 423 in rodentneuronal cell lines include the high molecular weight neurofilamentprotein in an aggregated state, but do not exclude the possibility thatthey also include altered MAPs. We refer to them as presumptive-NFHaggregates (pNFH). Dose-dependent inhibition of pNFH aggregates in thecytoplasm could be demonstrated with thionine in untransfected PC-12cells.

Example 7 Transfection of Neuronal Cell Lines with Full-length andTruncated Tau Proteins and Effects of Tao Aggregation Inhibitors

A. PC-12 Cells

PC-12 cells were transfected with the pIF2 vector containing either thePHF-core tau fragment truncated at Glu-391 or full-length tau protein.As with 3T3 fibroblasts, no viable cell lines transfected with truncatedtau were produced unless cells were grown in thionine followingtransfection. Once stahilised, transfected cell lines were analysed inthe presence or absence of db-cAMP and in the presence and absence ofthionine. Two end-points were examined: formation of cytoplasmic pNPHaggregates, and distribution of pNFH immunoreactivity into neurites.

Brief incubation with db-cAMP increased the proportion of cellscontaining neurofilament aggregates from 9% to 37% (p<0.001). Thiseffect was seen both in cells trarisfected with truncated tau (10% vs47%, p<0.001), and the differential effect of truncated tau was itselfsignificant (p=0.005). Thus, transfection with truncated tau accentuatedthe formation of pNFH aggregates in response to db-cAMP.

The effect of withdrawal of thionine after db-cAMP treatment was todouble the frequency of cells with pNFH aggregates (27% vs 49%, p=0.05).These increases were seen for cells transfected with both full-lengthtau (16% vs 32%) and truncated tau (36% vs 60%). A further effect wasthionine-dependent incorporation of pNFH immunoreactivity into neurites.This was particularly evident in PC-12 cells transfected with truncated,but not full-length tau or untransfected cells (pNFH-neurite indices0.49 vs 0.04 with and without thionine respectively, p=007).

B. NIE-115 Cells

In general, pNFH aggregates seen in the cytoplasm of N2A 20 cells didnot occur in untransfected NIE cells. Rather, pNFH immunoreactivity wasnormally incorporated into growing neurites during the course ofdifferentiation, although an early perinuclear-arc stage was also seen.NIE cells were transfected as above with the pIF2 vector containingeither full-length or truncated tau protein and grown in the presence ofthionine. The effects of adding db-cAMP in the presence or absence ofthionine were then examined.

As with PC-12 cells, no stable NIE cells transfected with truncated tauwere produced in the absence of thionine. Those transfected withtruncated tau produced a significantly higher overall frequency of pNFHaggregates in the cytoplasm than cells transfected with full-length tau(9% vs 26%, p<0.001), and incubation with db-cAMP induced pNFHaggregates in cells transfected with truncated tau but not infull-length tau transfectants (6% vs 36%, p<0.001).

In cells transfected with full-length tau, the presence of thionine didnot interfere with the incorporation of transgenic tau protein into themicrotubular cytoskeleton, including the microtubule organising centre,diffuse cytoplasmic distribution and extension into neurites. Withdrawalof thionine in cells transfected with full-length tau increased theproportion containing pNFH aggregates (7% vs 16%, p=0.03), In cellstransfected wilh truncated tau thionine withdrawal resulted in increasedpNFH aggregates in specific cell lines (e.g. NIE-ND6, 14% vs 44%,p=0.07), which were also characterised by suppression ofdifferentiation. This revision to a phenotype previously seen only inundifferentiated N2A cells, but not in NIE cells was striking.

As with PC-12 cells, thionine-dependent incorporation of pNFH intoneurites could be demonstrated after db-cAMP treatment in certain cells(e.g. NIE-ND1, pNFH-neurite indices 0.1 vs 0.66 with and withoutthionine respectively, p=0.01). Thionine-dependent transport of pNFHinto neurites could be seen quantitatively as a reversal of therelationship between cytoplasmic and neuritic neurofilarnent NHFimmunoreactivity in transfected cell in the presence of thionine(r=−0.52 vs r=+0.52 without and with thionine; p=0.01 and 0.02.respectively).

1. A method for the prophylaxis of Alzheimer's disease, motor neuronedisease, Lewy body disease, Pick's disease, or progressive supranuclearpalsy, which method comprises administering to a subject in need thereofan effective amount of an agent which modulates or inhibits tau-tauassociation and which does not inhibit tau-tubulin binding.
 2. A methodaccording to claim 1 for the prophylaxis of Alzheimer's disease.
 3. Amethod according to claim 1 wherein said agent is phenothiazines of theformulae:

wherein: R₁, R₃, R₄, R₆, R₇, and R₉ are independently selected fromhydrogen, halogen, hydroxy, carboxy, substituted or unsubstituted alkyl,haloalkyl or alkoxy; R₅, each R₁₀ and each R₁₁ are independentlyselected from hydrogen, hydroxy, carboxy, substituted or unsubstitutedalkyl, haloalkyl or alkoxy; and pharmaceutically acceptable saltsthereof.
 4. A method according to claim 3 wherein: R₁, R₃, R₄, R₅, R₆,R₇, R₉, each R₁₀ and each R₁₁ are independently selected from hydrogen,—CH₃, —C₂H₅ or —C₃H₇.
 5. A method according to claim 3 wherein saidagent is a phenothiazine of the formula (I):

wherein: R₁, R₃, R₄, R₆, R₇, and R₉ are independently selected fromhydrogen, halogen, hydroxy, carboxy, substituted or unsubstituted alkyl,haloalkyl or alkoxy; R₅, R₁₀ and R₁₁ are independently selected fromhydrogen, hydroxy, carboxy, substituted or unsubstituted alkyl,haloalkyl or alkoxy; and pharmaceutically acceptable salts thereof.
 6. Amethod according to claim 5 wherein R₅ is hydrogen.
 7. A methodaccording to claim 1, wherein said compound is selected from the groupconsisting of: Toluidine Blue O, thionine, Azure A, Azure B, and1,9-dimethyl-Methylene Blue.
 8. A method for the prophylaxis ofAlzheimer's disease, motor neurone disease, Lewy body disease, Pick'sdisease, or progressive supranuclear palsy, which method comprisesadministering to a subject in need thereof an effective amount ofMethylene Blue.
 9. A method according to claim 8, for the prophylaxis ofAlzheimer's disease.